1. Field of the invention
This invention relates to a means for electrophoresis comprising an aqueous polyacrylamide gel which is suitably employable for analysis of biopolymers such as proteins, as well as for determination of base sequence of DNA, RNA, their fragments, and their derivatives, and more particularly relates to an improvement of a membrane (slab or layer) for electrophoresis having an improved molecular sieve function over a wide range, that is, from a low molecular weight portion to a high molecular weight portion.
2. Description of prior art
Recently, electrophoresis has been frequently used for the analysis of biopolymers such as proteins, or for determination of base sequence of DNA or RNA.
As the membrane or sheet for the electrophoresis, a filter paper was previously employed, but recently an agarose gel membrane or a polyacrylamide gel membrane (or medium) has been employed from the viewpoints of their advantageous properties. Particularly, the polyacrylamide gel membrane showing a molecular sieve function and a function to separate, fractionate, analyse or assay a high molecular weight portion of proteins originating from a living body is widely used. More particularly, in the method for determination of base sequence of DNA, RNA, their fragments, and their derivatives according to a post-label method, a procedure of slab electrophoresis using a polyacrylamide gel membrane has become essential.
In the case that the analysis of proteins or determination of base sequence of DNA, RNA, their fragments is performed utilizing difference of their molecular weights in a run of electrophoresis, it is known that a polyacrylamide gel membrane having a uniform thickness gives a wide band interval in a low molecular weight portion and a narrow band interval in a high molecular weight portion. Thus, the separation of proteins, or DNA or RNA is not satisfactory in the high molecular weight portion. Accordingly, a polyacrylamide gel membrane which has an improved molecular sieve function, i.e., up to a high molecular weight portion, is required. A polyacrylamide gel membrane having a polyacrylamide gel concentration gradient or a buffer solution concentration gradient in the direction of electrophoresis is already used to analyze proteins equally over a wide range of molecular weight part. Japanese Patent Provisional Publication No. 60(1985)-235819 (corresponding to European Patent Provisional Publication No. 0159694A) describes a process for the preparation of polyacrylamide gel membrane for electrophoresis having a polyacrylamide concentration (i.e., gel concentration or pore size) gradient (gradient gel membrane) and an apparatus thereof. The polyacrylamide gel membrane can be prepared by closslinking polymerization of an aqueous solution containing an acrylamide and a crosslinking agent in the form of a thin layer formed on a surface of a support using an ionization rays such as an electronic beam to give a concentration gradient in the gel membrane. However, it is extremely complicate to manufacture the apparatus and to control the electronic beam. Further, the gradient gel membrane has problems of its mass scale production such that the preparation thereof requires a long period of time, reproducibility is insufficient, and preparation thereof frequently failed. Furthermore, the gradient gel membrane is apt to deform when it is dyed to visualize the segregated and fractionated image, because the degree of swelling is affected by the polyacrylamide gel concentration.